Downregulation of hepatic ecto‐5′‐nucleotidase (CD73) in a mouse model of alcoholic liver injury and in patients with alcoholic steatohepatitis

Abstract

Chronic liver diseases are a significant and growing medical problem in need of therapeutic advances. Strategies with the greatest potential to contribute broad impacts are those that can block the disease process early, and independently of the underlying etiology. The enzyme ecto‐5′‐nucleotidase (CD73), which metabolizes extracellular adenosine monophosphate (AMP) to adenosine, represents a promising novel liver disease target. Previous studies with CD73−/− mice suggested that CD73 significantly contributes to various forms of liver injury, including alcoholic steatosis. However, the functional and regulatory mechanisms of CD73 during liver injury are poorly understood. The objective of this study was to determine the dynamics of CD73 regulation in response to ethanol in vivo and ex vivo, and to compare these changes to human ASH patients.MethodsEthanol administration was performed using the Lieber DeCarli method in female C57BL/6J mice (14–16 weeks of age), with 1 or 2 week duration of ethanol diet, and a final concentration of 3.3% or 5% vol/vol ethanol (n=4–8 mice per group, 3 independent studies). Freshly isolated sandwich‐cultured hepatocytes from female C57BL/6J mice were treated with ethanol (50 or100mM for 3h or 24h), followed by RNA isolation, quantitative real‐time PCR and Western blot. CD73 gene expression analysis was performed on biopsy specimens from human ASH patients. Biochemical and histological assessment of CD73 expression and enzymatic activity was performed on liver lysates, cryosections, and isolated mouse hepatocytes using commercially available assays, western blotting, and enzyme histochemistry procedures.ResultsCD73 protein expression in the livers of mice receiving high dose of ethanol were significantly downregulated, by 32% (p<0.05) after one week, and 51% (p<0.01) after two weeks, with concomitant decrease in cell surface AMPase activity. This effect was milder in the group receiving the lower ethanol dose, where no changes were observed after 1 week, and 20% reduction in CD73 expression was seen after 2 weeks. Expression levels of CD73 mRNA in biopsy specimens from patients with ASH (n=10) were downregulated to 58+11.1% compared to control human livers (n=2). In contrast, ethanol treatment of isolated mouse hepatocytes caused a transcriptional CD73 upregulation and a significant redistribution of cell surface AMPase activity, demonstrating that CD73 is a target of the ethanol effects ex vivo.ConclusionCD73 expression and enzymatic activity are downregulated in response to ethanol in mouse ethanol‐treated livers and in patients with alcoholic liver disease. Since CD73 downregulation in response to ethanol in the mouse model occurs early (i.e. prior to the development of steatosis), our findings point to possible non‐enzymatic pathways for CD73‐dependent signaling during chronic alcohol‐induced liver injury. Additional mechanistic studies are needed to reconcile the ex vivo and in vivo effects of CD73 to better understand its regulation and signaling function during hepatocyte injury and liver disease.Support or Funding InformationSupported by NIH grants DK093776 and DK102450